Veterinary World, EISSN: 2231-0916

Available at www.veterinaryworld.org/Vol.6/Oct-2013/10.pdf

RESEARCH ARTICLE
Open Access

Molecular epidemiology of canine parvovirus in southern India

V. M. Vivek Srinivas1, H. K. Mukhopadhyay1, J. Thanislass2, P. X. Antony1 and R. M. Pillai1
1. Department of Microbiology, Rajiv Gandhi College of Veterinary and Animal Sciences,
Puducherry - 605 009, India; 2. Department of Veterinary Biochemistry, Rajiv Gandhi College of Veterinary
and Animal Sciences, Puducherry - 605 009, Iindia
Corresponding author: V. M. Vivek Srinivas, Present address: FMD Vaccine Production Lab, Indian Veterinary Research
Institute (IVRI), Hebbal, Bangalore-560 024. Ph: +91 97428 91992, email: vivekvet24@gmail.com
Received: 29-05-2013, Revised: 02-07-2013, Accepted: 03-07-2013, Published online: 13-08-2013
doi: 10.14202/vetworld.2013.744-749
How to cite this article: Srinivas VMV, Mukhopadhyay HK, Thanislass J, Antony PX and Pillai RM (2013) Molecular
epidemiology of canine parvovirus in southern India, Veterinary World 6(10): 744-749.

Abstract
Aim: The present study was conducted to isolate and characterize canine parvovirus circulating in Southern India by genetic
analysis of VP2 capsid protein gene.
Materials and Methods: In this study, 128 samples were collected from nine different locations covering five Southern
Indian states (Pondicherry, Tamil Nadu, Kerala, Andhra Pradesh and Karnataka) . Out of 128 samples, 69 samples were found
to be positive by PCR assay. Out of 69 positive samples, 36 were randomly selected and processed for virus isolation. Twenty
viruses could be isolated successfully and 18 randomly selected isolate were subjected to VP2 gene sequence analysis along
with 6 random clinical samples.
Result: Seventeen isolates and 5 clinical samples were characterized as New CPV-2a (CPV2a with 297-SerAla). But one
isolate and one clinical sample had amino acids variations which were characteristics of New CPV-2b. The phylogenetic
analysis revealed that one of the field isolates was found to be phylogenetically closely related to New CPV-2b strains of India;
rest other sequences was found to share ancestral origins with New CPV-2a reference strains of Japan, China, Thailand and India.
Conclusion: The present study revealed that the predominant CPV strain circulating in Southern India is New CPV-2a. There
is also enough indication of New CPV-2b strain from different states of Southern India.
Keywords: Canine parvovirus (CPV), crandell feline kidney (CRFK) cell line, molecular epidemiology, phylogenetic
analysis, sequence analysis.
Introduction

Canine parvovirus (CPV) is the most significant

viral cause of acute haemorrhagic enteritis and
myocarditis in puppies over the age of 3-4 months [1].
CPV presumably originated as a host range variant
from feline panleukopenia virus (FLV) that adapted to
new canine host via wild carnivores like minks and
foxes [2]. A small, round non-enveloped virus was
observed by electron microscopy in stool specimens
and tissues in affected animals. Subsequently, a novel
parvovirus was isolated in both canine and feline cell
cultures [3]. The Canine parvovirus 2 (CPV-2) belongs
to the family Parvoviridae with the genome of linear
single stranded DNA of 5.2 kb in size. The CPV
genome has two open reading frames (ORF) of which
the first ORF encodes one non structural protein (NS1)
and the second ORF encodes two capsid proteins (VP1
and VP2) which are translated from alternatively
spliced mRNA [4]. VP2 gene mainly comprises the
icosahedral capsid of CPV, and only a few amino acid
substitutions in its sequence can alter relevant
immunological characteristics of the virus [5, 6, 7].
Canine parvovirus (CPV) caused by CPV-2
Copyright: The authors. This article is an open access article licensed
under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0) which permits
unrestricted use, distribution and reproduction in any medium,
provided the work is properly cited.

Veterinary World, EISSN: 2231-0916

emerged in 1978 as a highly contagious and very

serious disease in dogs [1]. But CPV-2 underwent
evolutionary changes after its emergence in the late
1970s and within few years CPV-2 has been globally
replaced by the newer antigenic variants viz., CPV-2a
and CPV-2b [1, 4, 8]. At present, the variants of CPV
are distributed worldwide and the original prototype 2
no longer circulates in the dog population [9, 10, 11].
In 2000, a new variant of CPV which was named as
CPV-2c was reported in Italy [12]. This new variant
was distinguishable from CPV 2a/2b by the
substitution of Glu in lieu of Asn/Asp at 426 residue of
the VP2 capsid protein; therefore it was also referred to
as Glu-426 mutant [12]. Additional amino acid
difference was observed in both CPV-2a and CPV-2b at
position 297 (Ser to Ala) [10]. This mutation was first
appeared in 1993 in German CPV isolates and was
designated as New CPV-2a/2b [10].
Molecular diagnostic techniques like PCR based
methods had been the most reliable techniques having
high degree of sensitivity and specificity in detecting
CPV from faecal samples [12]. Amplification of VP2
gene fragment (capsid protein) using primers like
Hfor/Hrev and subsequent sequencing of the PCR
products covering the informative amino acids would
definitely help in detecting genetic variation which
exists between CPV-2 and its variants [12].
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Most commercial vaccines available in India contain

CPV-2 strain whereas; only one CPV-2b based vaccine
is available [13]. Therefore, further epidemiological
survey and molecular characterization of CPV
involving samples from larger geographical area
would be extremely helpful in formulating a suitable
vaccination strategy.
Hence, a study was planned on molecular survey
and characterization of canine parvovirus in Southern
India using PCR and subsequent sequencing of the
PCR products covering a portion of VP2 gene (capsid
protein). The genetic analysis of canine parvovirus
strains in dog population of various states in Southern
India will reveal the predominant CPV strain/strains
responsible for causing outbreaks in a larger
geographical area.
Materials and Methods
Clinical samples: A total of 128 faecal samples/rectal
swabs were collected from the dogs suspected to be
suffering from diarrhoea/enteritis due to canine
parvovirus infection from nine different cities namely
Puducherry (Pondicherry), Chennai, Coimbatore
(Tamil Nadu), Hyderabad, Tirupati (Andhra Pradesh),
Bangalore (Karnataka), Thrissur, Palakad and
Trivandrum (Kerala) covering all the Southern Indian
states, India, during a period of 11 months from April
2011 to February 2012 (Table-1). The collected
samples were emulsified in 1 ml of 0.1 M PBS of pH
7.4 and centrifuged at 6000 rpm for 15 min at 4oC. The
supernatant was collected and used for PCR
amplification and for virus isolation [13].
Template DNA preparation: Hundred microlitres of the
processed supernatant was used for template DNA
preparation by boiling at 96oC for 10 min and chilling
immediately in crushed ice [14]. The supernatants were
diluted 1:10 in distilled water to reduce residual
inhibitors of DNA polymerase activity [15].
Primer pair and PCR amplification: The processed
samples were screened by primer pair H-for (5'
CAGGTGATGAAT TTGCTACA-3') and H-rev (5'CATTTGGATAAACTGGTGGT-3') that amplifies
630 bp fragment of the gene encoding capsid protein.
PCR amplification was carried out as per Buonavoglia
[12] and the products were analyzed by electrophoresis
using 1.5% agarose gel in Tris acetate EDTA (TAE)
buffer (1X).
Buonavoglia designed the primer pair Hfor/Hrev and
utilized them for sequencing studies of canine
parvovirus since this primer pair amplifies a portion of
VP2 gene containing the critical amino acids such as
297, 300, 305, 375 and 426, which helps in
characterization of canine parvovirus [12].
Virus isolation: Thirty six processed clinical samples,
representing all the nine different locations which were
positive by PCR are randomly selected were filtered
using 0.22 mm membrane filter (Millipore) and the
filtrates were used for virus isolation in 70-75% CRFK
Veterinary World, EISSN: 2231-0916

cell line monolayer. Virus isolation was carried out as

per the procedure recommended by Hirayama [16].
The infected monolayers were harvested at the 4th day
of post infection (with or without CPE) by three cycles
of alternative freezing and thawing. The virus
supernatants were screened for the presence of virus by
PCR using the same primer pair Hfor and Hrev.
Sequencing and phylogenetic analysis: The amplified
PCR products of 18 randomly selected cell culture
isolates and six randomly selected clinical samples
were gel extracted and custom sequenced with primer
pair Hfor/Hrev using the automated sequencer,
Applied Biosystem 3100. The specificity of the
sequences obtained, the nucleotide variations and
amino acid variations with respect to the VP2 gene
sequence of canine parvovirus were determined using
BLAST [Basic Local Alignment Search Tool]
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) [13]. The
nucleotide sequences of VP2 gene were aligned with
sequences of prototype CPV strains (M38246- FPV;
M38245-CPV-2; M24003-CPV-2a; M74849-CPV-2b;
AY742953-New CPV-2a; AY742955-New CPV-2b;
FJ222821- CPV-2c) using ClustalW (www.ebi.ac.uk/
clustalw) and analyzed for the nucleotide variation of
VP2 gene at positions 3675, 3685, 3699, 3909 and
4064 with the corresponding amino acid residues at
297, 300, 305, 375 and 426, respectively [13].
For phylogenetic analysis, 30 canine parvovirus
sequences from various parts of the world were
retrieved from the GenBank and used. The sequences
were aligned using ClustalW 1.8 program and .aln and
.nxs files were generated. The .aln file was converted to
.meg file using Mega4.1 [17] and Neighbor Joining
tree (NJ tree) was constructed (bootstrap replicates =
1000; seed = 64,248) using Kimura 2 parameter
method for pairwise deletion at uniform rates [13].
The CPV sequences of 18 cell culture isolates and
6 clinical samples under study were submitted in
GenBank under the accession numbers JF900758,
JF900759, JF900760, JF900761, JF900762,
JN008380, JN008381, JN008382, JN008383,
JN008384, JN008385, JN008386, JN008387,
JN008388, JN008389, JN008390, JN008391,
JN008392, JN008393, JN008394, JN008395,
JN008396, JN008397 and JN008398 (Table-1).

Results and Discussion

Out of 128 samples screened, 69 (53.90%)

samples were found to be positive by PCR assay using
H primer. The disease was predominantly noticed in
dogs between 0-6 months (73.91%) as also reported by
other authors [13, 18, 19]. It was well known that
increased intestinal epithelial turnover caused by
changes in the microflora, diet (weaning) and
diminishing maternal antibody level were the
predisposing factors to CPV infection in pups [20]. The
occurrence of CPV enteritis, in this study, was found to
be more in males (62.31%) in comparison to females
(37.68%). High percentage of occurrence of CPV
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Table-1. Details of occurrence of CPV based on location (southern India)
State

Place

Pondicherry
Tamil Nadu

Pondicherry
Chennai
Coimbatore
Thrissur
Palakkad
Trivandrum
Hyderabad
Tirupati
Bangalore

Kerala
Andhra Pradesh
Karnataka

No. of clinical
samples collected

No. of samples positive

by PCR assay (Hfor/Hrev)

25
20
10
10
10
10
10
15
18

11
12
8
4
6
3
7
9
9

128

69(53.90%)

Total

Sequences submitted in Genbank

(Accession numbers)
JN008387, JN008388, JN008389
JF900758, JN008383, JN008397
JN008384, JN008396
JN008386
JF900762*, JN008385
JN008392
JF900761, JN008393*, JN008398
JF900759, JN008390, JN008391
JF900760, JN008380, JN008381,
JN008382, JN008394, JN008395
24 Sequence

* Nucleotide variations were characteristics of New CPV-2b (CPV-2b with amino acid variation 297Ser Ala). Other Sequences were
characteristics of New CPV-2a (CPV-2a with amino acid variation 297 SerAla)

Table-2. Details of occurrence of CPV based on age, sex and vaccination status
Status
0-6 months
6-12 months
Above 12 months
Male
Female
Non-vaccinated
Vaccinated

No. of samples collected

No. of samples positive by PCR assay

88
29
11
77
51
96
32

51 (73.91%)
13 (18.84%)
5 (7.24%)
43 (62.31%)
26 (37.68%)
58 (84.05%)
11 (15.94%)

Table-3. Nucleotides and amino acid variation in the VP2 protein of different CPV-2
CPV Strains

Amino acid residue in VP2

CPV-2
CPV-2a
CPV-2b
New CPV-2a
New CPV-2b
CPV-2c

Nucleotides in CPV Genome

3676

3685

3699

3909

4062

297
TCT/Ser
TCT/Ser
TCT/Ser
GCT/Ala
GCT/Ala
GCT/Ala

300
GCT/Ala
GGT/Gly
GGT/Gly
GGT/Gly
GGT/Gly
GGT/Gly

305
GAT/Asp
TAT/Tyr
TAT/Tyr
TAT/Tyr
TAT/Tyr
TAT/Tyr

375
AAT/Asn
GAT/Asp
GAT/Asp
GAT/Asp
GAT/Asp
GAT/Asp

426
AAT/Asn
AAT/Asn
GAT/Asp
AAT/Asn
GAT/Asp
GAA/Glu

infection in non-vaccinated animals (84.05%)

indicated that current vaccines confer reasonably good
protection [2, 21], though there are few reports of
vaccinated animals coming down with CPV infection
indicating vaccine failure [13, 19, 20] (Table-2).
Isolation of virus was attempted in CRFK cell line
from 36 samples found positive by PCR assay
representing the diverse locations of collection.
Twenty samples showed mild cytopathic effects in the
form of rounding, increased granularity and detached
cells, from the third passage level onwards. Moderate
isolation rate (55.55%) in this study may be attributable
to the presence of antibodies in the intestinal lumen of
the infected dogs, which may bind virions and prevent
viral attachment to cell receptors. Similar observations
were also made by Decaro and Mohanraj who observed
that the isolation of CPV could be done only for few
days of post infection [13, 14]. Cryolysates of all the
twenty samples were confirmed positive by PCR assay
using Hfor/Hrev primer pair at the third passage level.
In this study, the amplified PCR products of
eighteen cell culture isolates and six clinical samples
were gel extracted and custom sequenced using
Automated Sequencer, Applied Biosciences 3100. The
sequences obtained were subjected to 'BLAST' to study
specificity and strain identity. Sequences obtained
were found to be highly specific to CPV as indicated by
Veterinary World, EISSN: 2231-0916

the maximum identity (95-99%) obtained with VP2

gene sequence of other canine parvovirus strains
available in the GenBank.
The partial VP2 gene of 24 sequences was aligned
with the reference strains for sequence analysis. In
comparison to prototype CPV-2 (CPV b, M38245), the
cell culture isolates and the clinical samples under
study had nucleotide variations at position 3675 (T
G); 3685 (C G); 3698 (G T); 3908 (A G) and
identical nucleotide (A) at position 4062 (Table 2).
Seventeen cell culture isolates and five clinical
samples had amino acids Ala, Gly, Tyr, Asp and Asn at
residues 297, 300, 305, 375 and 426 respectively.
These nucleotide variations were characteristics of
New CPV-2a (CPV-2a with nucleotide variation TG
at position 3675 or CPV-2a with amino acid variation
297-Ser Ala). In one cell culture isolate JN008393
(Hyderabad) and one clinical sample JF900762
(Palakkad) had amino acids Ala, Gly, Tyr, Asp and Asp
at residues 297, 300, 305, 375 and 426 respectively.
These nucleotide variations were characteristics of
New CPV-2b (CPV-2b with nucleotide variation TG
at position 3675 or CPV-2b with amino acid variation
297-SerAla).
Twenty two out of 24 sequences had a nucleotide
variations which were characteristics of New CPV-2a,
was also reported by other authors [10, 13, 15, 22]. This
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Figure-1. Alignment of the deduced amino acid sequences of partial VP2 gene. Sequence of the representative canine parvovirus field
isolate/sample JN008391, JN008395 (New CPV 2a), JN008393, JF900762 (New CPV-2b) is shown aligned with the reference strains M38246
(FPV), M38245 (CPV-2), M24003 (CPV-2a), M74849 (CPV-2b), AY742953 (New CPV-2a), AY742955 (New CPV-2b) and FJ222821 (CPV-2c)
obtained from the GenBank.

is in accordance with the result reported by Chinchkar,

who also indicated that, CPV-2a was the predominant
type in India [23]. They reported that out of twenty two
CPV isolates from different parts of India, eighteen
belonged to CPV-2a, remaining four belonged to CPV2b and sixteen of the twenty two isolates had
substitution 297-Ser Ala. Nandi also reported the
circulation of strain CPV-2a, CPV-2b and CPV-2c in
the northern Indian geographical area [24]. Similarly,
two New CPV-2b strains were detected indicating the
presence of New CPV-2b also in certain locations of
Southern India. Alignment of the deduced amino acid
sequences of partial VP2 gene of the representative
canine parvovirus field isolate/sample JN008391,
JN008395 (New CPV 2a), JN008393, JF900762 (New
CPV-2b) and the reference strains is shown in Figure-1.
In addition to the nucleotide variations at positions
3675, 3685, 3698, 3908 and 4062, two additional nonsynonymous mutations were observed in the canine
parvovirus sequences under study. At nucleotide
position 3756-3758 (amino acid residue 324),
variations at 3756 (T A) and 3757 (A T) were
observed in sequences JN008387, JF900758,
JN008397, JN008383, JN008384, JN008396,
JN008386, JF900762, JN008385 and JN008392. This
type of mutation was also reported in the CPV isolated
in China by Zhang [14]. Horiuchi revealed that the
residue 324 was prone to strong positive selection in all
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carnivores [25]. The residue 324 was adjacent to

residue 323, which affected canine transferrin receptor
(TfR) binding and, together with residue 93,
determined the canine host range [25]. Therefore, the
324 mutation was likely to have an effect on the
parvovirus host range. This mutation resulted in the
codon change from TAT ATT, indicating change in
amino acid residue from Tyr Ile. Other one was at
nucleotide position 4104 (amino acid residue 440),
where variation (AG) was observed in case of all the
sequences under study except JN008388, JN008389.
This mutation resulted in the codon change from
ACA GCA, revealing change in amino acid residue
from Thr Ala. Similar codon change (GCA) at
nucleotide position 4104 was also reported in the CPV
isolated in Northern America by Kapil [26]. Therefore
these two non-synonymous mutations indicated that
the CPV strains were under constant selection pressure
and were constantly evoluting which might lead to
evolution of newer CPV strains/variants in the future.
In addition to non-synonymous mutations, three
synonymous mutations were also observed in the
sequences under study. One was at nucleotide position
3824 (amino acid residue 346), where variation (G
A) was observed in sequences JN008388 and
JN008389. This mutation resulted in the codon change
from GAG GAA, both of them coded for Glutamic
acid (Glu). Similar codon (GAA) change at nucleotide
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position 3824 was present in prototype FPV and was

also reported in the CPV isolated in India by Chinchkar
and Mohanraj [13, 23]. Other one was at nucleotide
position 4074 (amino acid residue 430), where
variation (G A) was seen in JN008380, JF900760,
JN008381, and JN008382, was also reported in the
CPV isolated in Japan by Doki [27]. This mutation
resulted in the codon change from TTGTTA, both of
them coded for Leucine (Leu). Another one mutation
was seen at nucleotide position 4121 (amino acid
residue 445), where variation (TC) was observed in
JN008388, JN008389, JN008390, JN008391,
JF900759, JN008393, JN900761, JN008398,
JN008380, JF900760, JN008381, JN008394,
JN008395 and JN008382. This particular variation
changed the codon from ACT ACC, both of which
coded for the same amino acid Threonine (Thr). This
synonymous mutation was not observed in any of the
previously isolated CPVs except in CPVs isolated from
India [13, 23]. All these mutations further confirmed
sustained constant evolution of canine parvovirus in
the geographical region of South India. Therefore,
constant surveillance and monitoring involving larger
geographical areas are must to identify the nucleotide
variations resulting in change in amino acids leading to
any possible antigenic variation between vaccine virus
and field strains.

(NewCPV-2a, Taiwan), CPU72696 (NewCPV-2b, Taiwan), EF599096

(NewCPV-2a, South Korea), EF599097 (NewCPV-2b, South Korea),
GU380298 (NewCPV-2a, China), GU380299 (NewCPV-2b, China),
GQ379044 (NewCPV-2a, Thailand), FJ869124 (NewCPV2b,Thailand), DQ025997 (NewCPV-2a, France), DQ025992
(NewCPV-2b, France).

Phylogenetic analysis of nucleotide sequences of

CPV obtained in this study with thirty reference CPV
strains from various parts of the world using neighborjoining method with Bootstrap consensus tree (Fig.-2)
revealed that one of the field isolates JN008393 was
found to be phylogenetically closely related (with
bootstrap value of 80) to New CPV-2b strains of India.
The sequence JN008394, JN008398, JN008395,
JN008391 and JF900761 was closely related to New
CPV-2a strain from Japan. The sequence JF900762,
JN008385, JN008392, JN008386, JN008383,
JN008397, JF900758, JN008384, JN008396 and
JN008387 were clustered phylogenetically with New
CPV-2a from China and Thailand The remaining
sequence JN008390, JN008382 JN008381, JN008380
and JF900760 were phylogenetically related to New
CPV-2a strain from India. Phylogenetic analysis
revealed that one of the isolates was found to be
phylogenetically closely related to New CPV-2b
strains of India and most of the sequences under this
study were clustered together showing distinct lineage
but share ancestral origin with New CPV-2a reference
strains of Japan, China, Thailand and India.
Conclusion

In this study, New CPV-2a (297-Ser Ala) was

found to be the predominant strain prevalent in
different cities across five Southern Indian states/
Union territories. There was also enough indication of
circulation of New CPV-2b strain from different states
of Southern India. As the CPV strains were found to be
under immense selection pressure and realizing the
constant mutations happening in the field strains,
continuous monitoring and detection of genetic
variations and antigenic changes will be of utmost
importance to control CPV infections. As there were
reports of occurrence of CPV in vaccinated pups under
this study, cross antigenic evaluation of CPV vaccines
and field strains is needed for evaluating the real
efficacy of CPV vaccines available in the market. The
major share of CPV positive cases in this study
belonged to unvaccinated dog population, so there is a
need for improving public awareness about
vaccination of pets against CPV infection.
Authors contributions
Fig 2. Neighbour Joining with Bootstrap consensus tree (Mega-4.1)
constructed using canine parvovirus sequences under study and the
reference sequences M38246 (FPV, USA), M38245 (CPV-2,USA),
M24003 (CPV-2a, USA), M74849 (CPV-2b, USA), AY742953
(NewCPV-2a, USA), AY742955 (NewCPV-2b, USA), AJ007500 (CPV2a, Africa), AJ007497 (CPV-2b, Africa), DQ182627 (NewCPV-2a,
India), DQ182625 (NewCPV-2b, India), D78585 (NewCPV-2a,
Japan), AB054220 (NewCPV-2b, Japan), AY742938 (NewCPV-2a,
Germany), AY742934 (NewCPV-2b, Germany), FJ005258 (NewCPV2a, Italy), FJ005265 (NewCPV-2b, Italy), FJ222821 (CPV-2c, Italy),
D Q 3 4 0 4 3 4 ( N e w C P V- 2 a , B ra z i l ) , A B 0 5 4 2 1 5 ( N e w C P V2a,Vietnam),AB054224 (NewCPV-2b, Vietnam), CPU72698

Veterinary World, EISSN: 2231-0916

VMVS and HKM were involved in the design of this

research. The research was done by VMVS. HKM has
monitored all the activities being a supervisor. HKM
and JT have helped in sequence analysis and
phylogenetic tree analysis. All authors drafted and
revised the manuscript. All authors read and approved
the final manuscript.
Acknowledgements

The authors are grateful to the Dean, Rajiv

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Available at www.veterinaryworld.org/Vol.6/Oct-2013/10.pdf

Gandhi College of Veterinary and Animal sciences,

Kurumbapet, Pondicherry, India for providing
necessary facilities and fund to conduct this research.

15.

Competing interests

The authors declare that they have no competing interests.

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